|Publication number||US7323092 B2|
|Application number||US 10/653,398|
|Publication date||Jan 29, 2008|
|Filing date||Sep 2, 2003|
|Priority date||Oct 24, 1997|
|Also published as||CA2306791A1, CA2306791C, EP1025434A1, EP1025434A4, US6660149, US7578915, US20040040850, US20040040851, WO1999022228A1|
|Publication number||10653398, 653398, US 7323092 B2, US 7323092B2, US-B2-7323092, US7323092 B2, US7323092B2|
|Inventors||Barry L. Karger, Lev Kotler, Frantisek Foret, Marek Minarik, Karel Kleparnik|
|Original Assignee||Northeastern University|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (9), Non-Patent Citations (3), Referenced by (4), Classifications (24), Legal Events (4)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This application is a divisional of U.S. application Ser. No. 09/530,118 filed Apr. 24, 2000 now U.S. Pat. No. 6,660,149, entitled A MULTICHANNEL MICROSCALE SYSTEM FOR HIGH THROUGHPUT PREPARATIVE SEPARATION WITH COMPREHENSIVE COLLECTION AND ANALYSIS which claims priority from U.S. Provisional Patent Application No. 60/062,787, filed Oct. 24, 1997, the whole of which are hereby incorporated by reference herein.
Part of the work leading to this invention was carried out with United States Government support provided under a grant from the Department of Energy, Grant No. DE-FG02-90ER60985. Therefore, the U.S. Government has certain rights in this invention.
Following the use of most modern separation techniques, further treatment of the separated components of a sample is required to obtain more complete information about the nature of the components. For example, methods of functional genomics (e.g., differential display (Liang et al., Science 257:967-971, 1992), AFLP (Vos et al., Nucl. Acid Res. 23:4407-4414, 1995), etc.) produce a pattern of separated DNA fragments, but the products of differentially expressed genes have to be identified separately. As another example, methods to discriminate mutations such as constant denaturant capillary electrophoresis (CDCE) also require subsequent determination of the specific mutation (Khrapko et al., Nucl. Acid Res. 22:364-369, 1994). To perform such a multidimensional analysis, a high throughput preparative separation system capable of collecting comprehensively all components of the sample mixture would be desirable.
Current micropreparative techniques for purification and fraction collection generally use either chromatography or electrophoresis for separation of the sample components. Fully automated single column systems are available, allowing fractionation and collection of specific sample components per run (Karger et al., U.S. Pat. No. 5,571,398 (1996); Carson et al., U.S. Pat. No. 5,126,025 (1992)). When fractions from multiple lanes are required, e.g., of DNA fragments, slab gel electrophoresis can be used for the simultaneous separation of the samples, followed by manual recovery of the desired fractions from the gel. This process is slow, labor intensive and imprecise. In another analytical approach, DNA fragments can be collected onto a membrane using direct transfer electrophoresis (Richterich et al., Meth. Enzymol. 218:187-222 1993). However, recovery of the samples from the membrane is slow and difficult.
The invention is directed to a modular multiple lane or capillary electrophoresis (chromatography) system that permits automated parallel separation and comprehensive collection of all fractions from samples in all lanes or columns, with the option of further on-line automated sample analysis of sample fractions. At its most basic, the system includes a separation unit such as a capillary column having each end immersed in a buffer solution, the inlet end being immersed in a regular buffer tank and the outlet end being in connection with the appropriate multi-well collection device. The outlet end may also be connected to a sheath flow generator. The capillary column, which may or may not have an inner coating and may be open tube or filled with any of a variety of different separation matrices, is used for separation of mixtures of compounds using any desired separation technique. The term “capillary column” is meant to include a vessel of any shape in which a microseparation technique can be carried out. For example, other types of separation units, such as channels in a microchip or other microfabricated device, are also contemplated.
Depending on the separation method chosen, a sample mixture could be introduced into one or more separation lanes simultaneously, using an electric field, or pressure, vacuum, or gravitational forces. Fractions usually are collected regardless of the sample composition in fixed time intervals, preferably every few seconds, into, e.g., a multi-well plate with fixed well volume, preferably, e.g., 0.5-10 microliter or smaller. The multi-well plate has sufficient capacity to collect all possible fractions during a separation run. Determination of sample separation profile(s) is accomplished by monitoring, e.g., an optical characteristic of the sample components, for example, laser induced fluorescence, color, light absorption (UV, visible or IR), using on-column or on-lane detection. After the run is completed, the desired fractions are selected using sample profiles recorded during the separation experiment. Determination of sample separation profile and selection of fractions may also be achieved in a post-process procedure, where collected fractions are scanned in a separate optical device capable of registering a desired optical characteristic of the collected material. Fractions of interest are transferred to microtubes or standard microtiter plates for further treatment.
The multi-well fraction collection unit, or plate, is preferably made of a solvent permeable gel, most preferably a hydrophilic, polymeric gel such as agarose or cross-linked polyacrylamide. A polymeric gel generally useful in the system of the invention is an entangled or cross-linked polymeric network interpenetrated by a suitable solvent so that the final composition has the required physico-chemical properties, e.g., sufficient electric conductivity (for, e.g., CE systems), rigidity and dimensional and chemical stability, to serve as the collection unit of the system of the invention. The polymer may or may not be cross-linked and may be linear or branched. Examples of suitable materials include, e.g., agarose, polyacrylamide, polyvinylpyrrolidone, polyethyleneglycol or polyvinylalcohol, and copolymers or combinations thereof. Other suitable materials for a collection unit include electrically conductive plastic or assemblies of micelles. The pore size(s) of gel network pores can be established as appropriate by modulating parameters such as polymer type, concentration, cross-linking agents and polymerization conditions.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims, taken in conjunction with the accompanying drawings, in which:
The system of the invention will now be described in detail using as an example a system for the separation of fluorescently labeled DNA fragments by capillary electrophoresis; however, such a system can be applied to different sample materials, e.g., proteins, other biopolymers, and low molecular weight compounds, or can incorporate other separation methods, as well. Any method of separation could be employed, including but not limited to capillary electrophoresis (CE), capillary isoelectric focusing (CIEF), capillary electrochromatography (CEC) and capillary liquid chromatography (CLC). Furthermore, any detection parameter would be useful in the method of the invention, such as, e.g., laser induced fluorescence, color, light absorption (UV, visible or IR), radioactivity or conductivity.
A basic micropreparative fraction collection system consists of the following main sections, as shown in
The preferred collection unit is an array of wells, each well capable of holding a fixed volume, e.g., ˜1 μL, that is formed in a medium such as glass, plastic, polymer or gel plate. For example, an array plate with 5,400 wells per channel would be capable of collecting fractions for up to 1.5 hours with zone resolution of 1 sec. In general, the collection unit, preferably capable of collecting fractions having very small volumes, is constructed so as to maintain fraction solvent evaporation at a very low level. For separation methods that use an electric field, the fraction collection unit is in electrical contact with the separation channels. In addition, the collection unit is preferably biocompatible and disposable.
Two different detection systems are contemplated in the preferred system for identification of the desired fractions in the wells. In the first option, as shown in
The second option, shown in
For repetitive use of the capillary array, e.g., for CE, replacement of the separation matrix may be necessary. Referring to
For collection of the individual zones as they exit the separation capillaries, either a liquid sheath or electrokinetic means can be used for completion of the electrical circuit for those separation methods requiring a circuit. In the first case, as shown in
When very small sample volumes are being handled, there are serious issues, such as rapid solvent evaporation, for fraction collection unit design. Therefore, the preferred multi-well collection unit of the invention is constructed of an electrically conductive biocompatible, solvent permeable material such as agarose or polyacrylamide gel. Forms for collection gel plate casting can easily be made, either by regular machining or by micromachining technologies. The collection plates may contain a large number of structures for sample collection (wells, channels) and also for further sample handling—desalting, filtration, enzyme reactions, etc. Similar gel based plates can also be used for preseparation sample treatment, as a sample application unit (10) (see
Examples of the system of the invention, including a gel based plate for collection of zones exiting the separation capillaries, are shown in
The zones exiting the capillaries are collected into the micro-wells on the gel plate; the wells may contain a collection fluid. Once collected, the fractions in the wells can be transferred out of the wells or processed directly in the wells. The evaporation of the liquid from the wells, which is a major problem in handling of minute sample volumes, can be reduced or eliminated in this case since the gel itself contains a large excess of water. For especially small volumes, the gel plate is partially immersed in a solvent bath so that positive liquid flow into the gel and the wells of the gel will be maintained. Gel plates could be cast with microchannels, allowing consecutive microfluidic sample handling. These plates may also be used to perform two dimensional electrophoresis or be utilized as a micro-storage device.
Beyond the use described above as a material from which microtiter plates can be made, solvent permeable, e.g., hydrophilic, gels are useful in many different ways, such as for fabrication of miniaturized devices for sample treatment, reaction and analysis. Microfabricated analytical devices are currently produced from standard solid materials such as glass, certain metals, silicon, silicon resins and other plastic materials. These materials are generally rigid and impermeable to both ions and water. Electric conductivity is provided only when metal or semiconductor materials are used. While the above mentioned materials can be used for fabrication of very small features such as channels for sample delivery and separation, sample inlet and outlet ports, unions, etc., some other desirable features such as permeability for water or selective permeability for ionic species and/or a low absorptivity surface cannot easily be achieved in prior art devices. Miniaturized devices fabricated of the solvent permeable gel material of the invention can contain all the desirable features of the devices of the prior art and in addition solve the problem of rapid evaporation of samples.
A miniaturized analogue of the microtiter well plate can easily be produced by gel casting into any desired shape. Referring to
The angle of capillary exit end orientation in relation to the opening of individual wells is an important parameter for ease of sample collection. Orientation at an angle, as shown in
Since the properties of the gel can easily be modified by changing gel concentration, crosslinking or chemically modifying the gel, functions difficult to incorporate with standard materials may be possible. For example, electrophoretic separation can be performed in a gel with an array of wells and the separated substances can easily be removed from the wells without tedious extraction from the gel. Pore and/or pH gradient gels would be especially beneficial for this application, e.g., for protein preparation. For example, Immobilin™ can be used as a gel matrix for micropreparative isoelectric focusing. Of course, other functionalized gels may be used. For example, immobilized antibody or antigen containing gels may be used for affinity capture. Since channels and wells of practically any shape can be easily fabricated by gel casting, many structures fabricated in “classical chips” can be fabricated in gel more cost effectively. In addition, enzymes can be immobilized in the gel structure, and reactions such as digestion (protein or DNA), PCR and sequencing can be carried out. The gel can be heated, e.g., by microwaves, if necessary. If the enzymes (substrates, template, . . .) are immobilized in the gel, little or no sample cleanup would be necessary compared to other sample handling systems. SSDNA can be fixed in the gel for specific hybridization to a complementary DNA strand. In addition, other biospecific groups such as antibodies could also be immobilized in individual wells. In particular, inert particles, such as beads, can be placed in individual wells, as carriers of active materials, e.g., antibodies, enzymes, substrates, etc. For, example, functionalized solid phase particles would be useful for on-plate combinatorial chemical analysis.
Besides bare gel blocks casted or molded for the purposes described above, other contemplated uses for hydrophilic gels are as components of “cassettes.” Such cassettes could be hybrid gel-plastic or gel-glass or gel-metal devices or chips, where a mold serves as a gel plate enclosure. All surfaces of the gel block would be covered, besides channels and wells. This design would both prevent excessive losses of water from the gel during sample manipulation (e.g., microwave heating) and ease the handling of the gel devices. A mold would be made as a reusable device, which would significantly reduce costs, especially if the mold contains embedded contacts or heating elements.
The following example is presented to illustrate the advantages of the present invention and to assist one of ordinary skill in making and using the same. These examples are not intended in any way otherwise to limit the scope of the disclosure.
To verify that individual components of a sample can be collected using the system of the invention with a microtiter multi-well collection plate, an experiment was conducted with a fluorescently labelled double stranded DNA restriction fragment mixture (commercially available as pBR322/Hinf I) as a sample. The mixture was separated by capillary electrophoresis (CE) in a 75 im i.d. polyvinylalcohol coated fused silica capillary filled with linear polyacrylamide (4% solution in 50 mM Tris/TAPS buffer) in an electric field of 370V/cm. The total capillary length was 27 cm and the length from injection to detection point was 25 cm. Injection was performed electrokinetically for 2-3 seconds at 370 V/cm. Detection was accomplished on-column by laser induced fluorescence using an argon ion laser (488 nm) and emission at 520 nm by means of confocal detection. The microtiter gel collection plate was a 3% agarose composite (a mixture of 1.5% large pore and 1.5% narrow pore agarose material), containing a single lane of microwells. During separation, the capillary was moved from one microwell to another in constant time intervals of 30 seconds. After deposition, the fractions were transferred out of the microwells, desalted and identified by re-injection and capillary electrophoresis.
While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US3912610 *||Jul 23, 1974||Oct 14, 1975||Icl Scient||Method for electroquantitative determination of proteins|
|US4716101 *||Dec 18, 1984||Dec 29, 1987||Calgene, Inc.||Rapid enzyme assay by product selective blot|
|US5143646 *||Jan 16, 1990||Sep 1, 1992||Fmc Corporation||Polysaccharide resolving gels and gel systems for stacking electrophoresis|
|US5541420||Dec 21, 1994||Jul 30, 1996||Hitachi, Ltd.||Multi-sample fraction collector by electrophoresis|
|US5800691 *||Dec 23, 1996||Sep 1, 1998||Guest Elchrom Scientific Ag||Electrophoresis gels containing sample wells with enlarged loading area|
|US5942443 *||Jun 28, 1996||Aug 24, 1999||Caliper Technologies Corporation||High throughput screening assay systems in microscale fluidic devices|
|US5944971 *||Apr 23, 1997||Aug 31, 1999||Lockheed Martin Energy Research Corporation||Large scale DNA microsequencing device|
|WO1996018891A1 *||Dec 15, 1994||Jun 20, 1996||University College London||Gel-matrix electrophoresis|
|WO2000050644A2 *||Feb 25, 2000||Aug 31, 2000||Mosaic Technologies||Methods for purifying dna using immobilized capture probes|
|1||Boss et al., "Multiple Sequential Fraction Collection of Peptides and Glycopeptides by High-Performance Capillary Electrophoresis", Analytical Biochemistry, vol. 230, pp. 123-129, (1995).|
|2||Irie et al., "Automated DNA Fragment Collection by Capillary Array Gel Electrophoresis in Search of Differentially Expressed Genes", Electrophoresis 21:367-374, (2000).|
|3||Weinmann et al., "Capillary Electrophoresis-Matrix-Assisted Laser-Desorption Ionization Mass Spectrometry of Proteins", Journal of Chromatography, vol. 680, pp. 353-361, (1994).|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US8263389||Jun 30, 2010||Sep 11, 2012||Biorep Technologies, Inc.||Perifusion device|
|US8785178||Jan 12, 2007||Jul 22, 2014||Biorep Technologies, Inc.||Perifusion device|
|US8865427||Aug 27, 2012||Oct 21, 2014||Biorep Technologies, Inc.||Perifusion device|
|US20080168847 *||Jan 12, 2007||Jul 17, 2008||Biorep Technologies, Inc.||Perifusion device|
|U.S. Classification||204/606, 204/470, 204/616|
|International Classification||C12M1/00, G01N1/10, C12N15/09, G01N30/80, G01N27/447, B01D15/08, G01N30/82, G01N27/453, G01N30/46|
|Cooperative Classification||B01D15/08, G01N30/466, G01N30/80, G01N30/82, G01N27/44717, G01N27/44782, G01N27/44791|
|European Classification||G01N27/447C7, G01N27/447B3, G01N27/447C5, G01N30/46E, G01N30/80|
|Jan 12, 2010||CC||Certificate of correction|
|Sep 5, 2011||REMI||Maintenance fee reminder mailed|
|Jan 29, 2012||LAPS||Lapse for failure to pay maintenance fees|
|Mar 20, 2012||FP||Expired due to failure to pay maintenance fee|
Effective date: 20120129